Coenzyme Q10 (2,3 dimethyl-5-methyl-6-decaprenyl benzoquinone) (“CoQ10”) levels in whole blood and plasma have been the subject of much inquiry as described, for example, in Tomasetti, M., Alleva, R., Solenghi, M. D., Littarru, G. P., Distribution of antioxidants among blood components and lipoproteins: significance of lipids/CoQ10 ratio as a possible marker of increased risk for atherosclerosis. BioFactors, 9, 231-240 (1999), the entire content of which is incorporated herein by reference. It is likely that plasma concentrations of CoQ10 reflect an overall metabolic demand, as discussed in Littarru, G. P., Lippa, S., Oradei, A., Fiorini, R. M., Mazzanti, L., Metabolic and diagnostic implications of human blood CoQ10 levels, in Biomedical and Clinical Aspects of Coenzyme Q vol. VI, (eds. K. Folkers, G. P. Littarru, T. Yamagami), Elsevier North Holland, pp. 167-178 (1991), the entire content of which is incorporated herein by reference. In addition, together with other lipophilic antioxidants, CoQ10 plays an intrinsic role in protecting circulating lipoproteins against oxidative damage. Therefore, the concentration of CoQ10 in lipoproteins and blood plasma could be of clinical importance regarding oxidative stress and antioxidant defense. Increased levels of CoQ10 enhance its antioxidant protection, even though the potential to act as an antioxidant in vivo probably depends not only on total CoQ10 concentration, but also on its redox status. The content of CoQ10 in single classes of lipoproteins has been found to be strictly correlated with CoQ10 plasma concentration. Previous reports have shown that the LDL-cholesterol/CoQ10 ratio significantly correlates with the total-cholesterol/HDL-cholesterol ratio which is usually considered a risk factor for atherosclerosis as described, for example in Alleva, R., Tomasetti, M., Bompadre, S., Littarru, G. P., Oxidation of LDL and their subfractions: kinetic aspects and CoQ10 content. Molec Asp Med, 18, s105-s112 (1997), the entire content of which is incorporated herein by reference. Some effective hypocholesterolemic agents, namely the statins, also lower plasma CoQ10 concentrations, owing to the common biosynthetic pathway of cholesterol and the isoprenoide side chain of coenzyme Q as described, for example, in Mortensen, S. A., Leth, A., Agner, E., Rohde, M., Dose-related decrease of serum coenzyme Q10 during treatment with HMG-CoA reductase inhibitors. Molec Asp Med, 18, s137-s144 (1997), the entire content of which is incorporated herein by reference. Therefore, it would be desirable to have an effective, reliable, fast method to measure CoQ10 concentrations in blood plasma or blood serum to monitor the CoQ10 levels in patients receiving hypocholesterolemic agents.
CoQ10 is used as a food supplement or as an adjunctive therapy in several diseases and the blood plasma or blood serum levels achieved upon oral administration of CoQ10 can correlate with clinical efficacy. Tests of blood plasma or blood serum levels of CoQ10 are useful for monitoring the bioavailability of orally administered coenzyme Q10.
Several methods have been described for assaying either total CoQ10 or the reduced (ubiquinol-10, CoQ10H2) and oxidized (ubiquinone-10) forms in blood plasma, and several of these methods are described in the following references, the entire contents of each of which are incorporated herein by reference: Lang, J. K, Packer, L., Quantitative determination of vitamin E and oxidized and reduced coenzyme Q by high-performance liquid chromatography with in-line ultraviolet and electrochemical detection, J Chromatogr, 385, 109-117 (1987); Finckh, B., Kontush, A., Commentz, J., Hubner, C., Burdeleski, M., Kohlschutter, A., High-performance liquid chromatography-coulometric electrochemical detection of ubiquinol 10, ubiquinone 10, carotenoids and tocopherols in neonatal plasma, in Methods in Enzymology, Vol 299, (Lester Packer Ed.), pp. 341-348, Academic Press, San Diego (1999); Podda, M., Weber, C., Traber, M. G., Milbradt, R., Packer, L., Sensitive high-performance liquid chromatography techniques for simultaneous determination of tocopherols, tocotrienols, ubiquinols and ubiquinones in biological samples in Methods in Enzymology, Vol 299, (Lester Packer Ed.), pp. 330-341, Academic Press, San Diego (1999); Finckh, B., Kontush, A., Commentz, J., Hubner, C., Burdeleski, M., Kohlschutter, A. Monitoring of ubiquinol 10, ubiquinone 10, carotenoids and tocopherols in neonatal plasma microsamples using high-performance liquid chromatography with coulometric electrochemical detection. Anal. Biochem, 232, 210-216 (1985); Okamoto, T., Fukunaga, Y., Ida, Y., Kishi, T., Determination of reduced and total ubiquinones in biological materials by liquid chromatography with electrochemical detection, J. Chromatogr, 430, 11-19 (1988); Grossi, G., Bargossi, A. M., Fiorella, P. L., Piazzi, S. Improved high-performance liquid chromatographic method for the determination of coenzyme Q10 in plasma. J. Chromatogr., 593, 217-226 (1988); Edlund, P. O., Determination of coenzyme CoQ10, α-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulometric and ultraviolet detection. J. Chromatogr, 425, 87-97 (1988); Lagendijk, J., Ubbink, J. B., Vermaak, W. J., Measurement of the ratio between the reduced and oxidized forms of coenzyme Q10 in human plasma as a possible marker of oxidative stress, J. Lipid Res, 37, 67-75 (1996); Yamashita, S., Yamamoto, Y., Simultaneous detection of ubiquinol and ubiquinone in human plasma as a marker of oxidative stress, Anal. Biochem, 250, 66-73 (1997). As used herein, ubiquinol means reduced CoQ10 and ubiquinone means oxidized CoQ10.
The previous methods of analyzing blood plasma to assay the concentration of CoQ10 have several disadvantages. Prior methods require that the CoQ10 be extracted from the plasma, followed by drying, which concentrates the extract. Losses in CoQ10 can occur during the drying and concentration step. In addition, these methods analyze the oxidized form of CoQ10, while most of the CoQ10 in the plasma is in the reduced form. These methods rely on oxidation of the CoQ10 during the extraction procedure. UV methods for assaying CoQ10 usually quantify the oxidized coenzyme at 275 nm. It is commonly assumed that CoQ10 is completely oxidized during the extraction and HPLC procedure, but this is not necessarily the case when the sample is fresh and the reduced form of CoQ10 largely predominates. Accordingly, these methods can result in underestimates of CoQ10 concentration if all of the CoQ10 is not oxidized.
The present invention is directed to a new, simplified method for evaluating total CoQ10 in blood plasma or blood serum. The method of the present invention results in reduced time and cost for the analysis of CoQ10 in plasma or serum as compared to prior methods, and provides more accurate results.